Methods for sequencing a biomolecule by detecting relative positions of hybridized probes

• US 9,702,003 B2
• Filed: 08/26/2014
• Issued: 07/11/2017
• Est. Priority Date: 11/16/2010
• Status: Active Grant

• Current Assignee: NABsys, Inc.
• Original Assignee: NABsys, Inc.
• Inventors: Goldstein, Peter H.
• Primary Examiner(s): Whisenant, Ethan C
• Application Number:
  US14/468,959
• Publication Number:
  US 20150045235A1
• Time in Patent Office: 1,050 Days
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2535/119 Double strand sequencing
• CPC Class Codes: C12Q 2565/107 Alteration in the property of hybridised versus free label oligonucleotides
• CPC Class Codes: C12Q 2565/631 being a biochannel or pore

Abstract

A sequencing method is presented in which a biomolecule is hybridized with a specially chosen pool of different probes of known sequence which can be electrically distinguished. The different probe types are tagged such that they can be distinguished from each other in a Hybridization Assisted Nanopore Sequencing (HANS) detection system, and their relative positions on the biomolecule can be determined as the biomolecule passes through a pore or channel. The methods eliminate, resolve, or greatly reduce ambiguities encountered in previous sequencing methods.

Claims

1. A method for determining a sequence of a biomolecule, the method comprising the steps of:

(a) identifying a set of k−

1-length subsequences that represent a plurality of substrings of a sequence string s of the biomolecule;

(b) for each of the k−

1-length subsequences, forming a pool of probes comprising four different k-mer extensions of the k−

1-length subsequence;

(c) for each pool formed in step (b);

(i) hybridizing the biomolecule with the four k-mer probes making up the pool; and

(ii) detecting relative positions of the k-mer probes that have attached to the biomolecule; and

(d) ordering the subsequences corresponding to the detected attached probes to determine the sequence string s of the biomolecule.

2. The method of claim 1, wherein each of the four k-mer probes in step (c) has a distinguishable tag attached, such that there are four different detectable tags used for a given pool of k-mers.

3. The method of claim 2, wherein step (c)(i) comprises hybridizing the biomolecule with all four of the k-mer probes making up the pool prior to detecting the relative positions of the attached k-mer probes in step (c)(ii), such that step (c)(ii) results in detecting the relative positions of all four of the k-mer probes making up the pool.

4. The method of claim 1, wherein step (c) comprises:

(A) hybridizing the biomolecule with two different k-mer probes selected from the four k-mer probes making up the pool, wherein the two selected k-mer probes have tags attached that are distinguishable from each other;

(B) following (A), where one or more binding events occur involving both of the selected k-mer probes, detecting relative positions of the two different k-mer probes that have attached to the biomolecule; and

(C) repeating (A) and (B) with another two different k-mer probes selected from the four k-mer probes making up the pool until hybridizations and detections are performed for all six pair combinations of the four k-mer probes making up the pool, thereby detecting the relative positions of all four of the k-mer probes making up the pool.

5. The method of claim 1, wherein step (c) comprises:

(A) hybridizing the biomolecule with a set of three k-mer probes selected from the four k-mer probes making up the pool, wherein the three selected k-mer probes have tags attached that are distinguishable from each other;

(B) following (A), where one or more binding events occur involving two or three of the selected k-mer probes, detecting relative positions of the two or three k-mer probes that have attached to the biomolecule; and

(C) repeating (A) and (B) with a different set of three k-mer probes selected from the four k-mer probes making up the pool until hybridizations and detections are performed for all four three-member combinations of the four k-mer probes making up the pool, thereby detecting the relative positions of all four of the k-mer probes making up the pool.

6. The method of claim 1, wherein step (c)(ii) comprises using HANS to detect the relative positions of the k-mer probes.

7. The method of claim 6, wherein step (c)(ii) comprises monitoring an electrical signal across a fluidic channel or pore or within a fluidic volume of a channel or pore as the hybridized biomolecule translocates therethrough, the electrical signal being indicative of hybridized portions of the biomolecule and non-hybridized portions of the biomolecule.

8. The method of claim 7, wherein the detected electrical signal allows differentiation between at least two of the k-mer probes hybridized to the biomolecule.

9. The method of claim 1, wherein step (c)(ii) comprises detecting an optical signal indicative of the relative position of at least two of the k-mer probes hybridized to the biomolecule.

10. The method of claim 1, wherein the set of k−

1-length subsequences represents all possible substrings of length k−

1 in the sequence string s.

11. The method of claim 1, wherein k is an integer from 3 to 10.

12. The method of claim 1, wherein s is a sequence string at least 100 bp in length.